KR20040015044A - Impartment of virus-resistance with the use of plant protein binding to plant virus transport protein - Google Patents

Abstract

Method for imparting plant virus-resistance to plants. Such methods include introducing into a plant cell a polynucleotide encoding a protein that binds to a plant virus transfer protein. In one embodiment the protein encoded by the above-described polynucleotide is selected by such sequence by substitution, deletion or addition of the sequence shown in 1- to 86-position of SEQ ID NO: 2 or one or several amino acids. It contains the derived sequence and binds to the moving protein of the plant virus. In one embodiment the above-described polynucleotides comprise a sequence shown in positions 14- to 271- of SEQ ID NO: 1 under stringent conditions or a nucleotide sequence capable of hybridizing with such nucleotide sequence.

Description

Immunity of virus-resistance with the use of plant protein binding to plant virus transport protein}

Some molds and bacteria invade plants by secreting cell wall-degrading enzymes. Plant viruses, such as tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), belonging to RNA viruses, on the other hand, do not encode the genes of cell wall-degrading enzymes, but only after entering through physical scars or transferred by insects or fungi. Can enter through the cell wall.

Highly infectious viruses, such as tobacco mosaic virus, exhibit massive proliferative activity, such as the production of 10 ⁶ offspring per day. This level of proliferation does not always cause disease. Initially infected cells are only part of the entire plant and most cells are not invaded. In many cases, the cells on the surface of the leaves are first infected with the virus and the virus migrates into the lobules, where the virus multiplies. The multiplied virus spreads into surrounding lobules. In this way, viruses are spread through plant tissues. This process of plant virus spread to neighboring cells is called cell-to-cell movement. It migrates to vascular bundle sheath, sieve parenchyma, and companion cells, and then begins to move from tissue to tissue via quadrant elements (Saibo-Kogaku [Cellular Engineering], Special Issue, Syokubuktu-Saibo-Kogaku). Plant Cellular Engineering series, Vol. 8, Shujyunsha, pp. 146-155, Chapter 3 "Uirius-Teikosei-no-tame-no-Senryaku [Stratagy for Virus Resistance]", p. 2, Yuichiro Watanabe, "Syokubutsu Uirusu-no-Saibokan-Iko [Cell-to-Cell Movement of Plant Virus] ").

Examples of host plants' resistant response to viral infection include: (1) absence of substantial disease signs (resistance) despite inhibition of virus growth or viral growth; (2) prevention of virus propagation only in the cell into which the virus first entered and migration of the virus to surrounding cells that prevent the virus from spreading throughout the plant (subliminal infection); (3) inhibition of virus propagation in infected leaves and long-distance migration from infected leaves to upper leaves; And (4) rapid necrosis of infection site tissue at an early stage of viral infection that does not cause any systemic infection. Virus is located on the spinal cord tissue or its periphery to avoid systemic infection (high sensitivity response). Efforts have been made to further explain the resistance mechanism based on these findings. With this effort, molecular studies of viral resistance have been carried out by physiological biochemical and genetic analysis in terms of viral infection and host plants (Saibo-Kogaku [Cellular Engineering], Special Issue, Syokubuktu-Saibo-Kogaku [Plant Cellular]). Engineering] series, Vol. 8, Shujyunsha, pp. 166-176, Chapter 3 "Uirius-Teikosei-no-tame-no-Senryaku [Stratagy for Virus Resistance]", p. 3, Hideki Takahashi, "Uirusu-ni- tai- suru-Syunkusyu-Teikosei [Host Resistance to Virus] ").

Proliferate through successive stages of infection, such as genomic replication within individual cells, cell-to-cell migration through plamodesmata, and long-distance migration through ducts (Carrington et al., (1996) Plant Cell 8, 1669-1681; Baker et al., (1997) Science 276, 726-733). In these stages, viral migration is facilitated by virus-encoded proteins called one or more transfer proteins (MPs) (Deom et al. (1002) Cell 69, 221-224). They must interact with various host factors (Carrington et al., Homology; Baker et al., Homology). Functional domains have been characterized within the MPs of many plant viruses. These include tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Two RNA binding domains in TMV have been identified within the C-terminal half of the MP (Citovsky et al., (1990) Cell 60, 637-647) whereas only one of these domains is the third C-terminal of the CMV MP. (3a protein; Vaquero et al., (1997) J. Gen. Virol. 78, 2095-2099). It is believed that this advantage of RNA binding forces the virus to migrate from cell to cell as a nucleoprotein complex (Lazarowitz and Beachy, (1999) Plant Cell 11, 535-548). Unlike the viral family, MPs form tubular structures (van Lent et al., (1991) J. Gen. Virol. 72, 2615-2623; Storms et al., (1995) Virology 214, 485-493; Huang et al., (2000) Virology 271, 58-64); Increasing the size exclusion limit of Plasmodesmata (Wolf et al., (1989) Science 246, 377-379). MPs have been found in connection with various subcellular structures, including endoplasmic reticulum, cytoskeleton and plasmoidesma (Tomenius et al., (1987) Virology 160, 363-371; Atkins et al., (1991) J Gen. Virol. 72, 209-211; Heinlein et al., (1995) Science 270, 1983-1985; McLean et al., (1995) Plant Cell 7, 2101-2114; Reichel et al., (1999). Trends Plant Sci. 4, 458-463).

Recently, host factors that interact with viral MPs have been of interest. Tm-2 and Tm-2 ² in tomatoes are reported as resistance genes for tomato mosaic virus (ToMV) (Hall, (1980). Euphytica 29, 189-197; Fraser (1990) Annu. Rev. Phytopathol. 28, 179-200. 25). Mutant virus strains that conquer the Tm-2 or Tm-2 ² phenotype have amino acid substitutions in the MP. This suggests an interaction between MP and resistant gene product (Meshi et al., (1989) Plant Cell 1, 515-522; Weber et al., (1993) J. Virol. 67, 6432-6438). Two homologous proteins in the Nicotiana tabacum and Arabidopsis thaliana Dan J family are identified in a yeast two-hybrid screen that interacts with tomato spot Wilt virus (TSMV) MP (Soellick et al., (2000) Proc. Natl. Acad. Sci. USA 97, 2373-2378). Pectin methylesterase located in Plasmodesmata was found to interact with TMV MP. This suggests that this enzyme induces MP and / or MP / RNA complexes into Plasmodesmata (Dorokhov et al., (1999) FEBS Lett. 461, 223-228; Chen et al., (2000) EMBO J 19, 913-920). CMV 2b protein (Ding et al., (1995) EMBO J. 14, 5762-5772), which is required for long-range viral migration, has been reported to interact with tobacco proteins similar to LytB, a progressive cellular protein involved in penicillin resistance in bacteria. (Ham et al., (1999) Mol. Cells 9, 548-555). Bringeti et al. (1998) EMBO J 17, 6739-6746 suggested that CMV 2b functions as an inhibitor of posttranslational gene silencing in host plants. Recently, Voinnet et al., (2000) Cell 103, 157-167, reported that potato virus XMP prevented the spread of gene silencing signals in N. benthamiana.

Thus there are many findings on plant virus MPs. Nevertheless, there are no reports on preventing plant virus infection and conferring viral resistance to plants.

Summary of the Invention

The inventors of the present invention cause the expression of a protein capable of binding to the plant virus mobile protein identified by the plant to be expressed in the plant, thereby preventing the plant virus mobile protein from interacting with the protein of the plant host against viral infection, thereby preventing the cells of the plant virus. Blocks two-cell migration and thus confers host plant resistance to plant viruses.

In one aspect, the invention relates to a method for imparting resistance to plant viruses to plants. The method consists in introducing a polynucleotide encoding a protein capable of binding to the plant protein of the plant virus into the cells of the plant.

In one embodiment the protein encoded by the polynucleotide comprises the sequence shown in positions 1 to 86 of SEQ ID NO: 2 or comprises a sequence having some amino acid substitutions, deletions and / or additions of plant viruses Binds to the moving protein.

In another embodiment the polynucleotide comprises the nucleotide sequence shown in positions 14-271 of SEQ ID NO: 1 under stringent conditions or a nucleotide sequence capable of hybridizing to the nucleotide sequence.

In one embodiment the plant virus is Tobamovirus , preferably Tomato Mosaic Virus (ToMV).

In one embodiment the polynucleotide is derived from Brassica campestris or Arabidopsis thaliana .

According to another aspect there is also provided a plant produced by the method of the invention.

In one embodiment the plant is a monocotyledonous or dicotyledonous plant. In one embodiment the plant is tobacco.

The present invention relates to a method for imparting plant virus resistance to plants. More preferably, the present invention relates to a method for imparting viral resistance to a plant by expressing a protein capable of binding to a plant virus transfer protein in the plant.

1 is an electrophoretic photograph showing the binding of ToMV MP to MIP102. (A) E. coli with pGEX-Bc2 plasmid was grown without IPTG (lane 1) or in the presence of IPTG. Total protein extracted from bacteria was electrophoresed in 10% SDS-polyacrylamide gel and stained with CBB. (B) MP binding was analyzed on restored protein blots. Protein analyzed in (A) was analyzed on PVDF membrane and probed with ³² P-labeled GST-PKA-MP. (C) Affinity-purified GST-MIP102 (lane 1) and GST (lane 2) were electrophoresed and stained on a 10% gel. Proteins analyzed in (C) were blotted onto PVDF membranes and analyzed with ³² P-labeled GST-PKA-MP as (B). The position of the molecular weight marker is shown on the left.

2 shows the nucleotide sequence of MIP102 cDNA and its putative amino acid sequence. The underlined amino acid sequences represent putative single-helix DNA binding domains used in the spirit of FIG. 3. Unique HindIII and EcoRI sites used for Southern blotting analysis are identified by double underlining. Arrows 1 and 2 represent the positions of the introns shown by genomic DNA analysis.

3 shows a number of sequence alignments and similar sequences of MIP102 and AtKELP in the GenBank database. The upper alignment shows the comparison of the putative amino acid sequence of MIP102 of K. Arabidopsis thaliana with KELP (AtKELP). ★ indicates matched amino acid residue between MIP102 and AtKELP. Subarrays represent comparisons of various proteins within putative single-helix binding domains. MIP102, AtKELP and KIWI were aligned with homologous sequences identified in other organisms such as humans (PC4 / p15) (Ge and Roeder, 1994; Kretzshmar et al., (1994) Cell 78, 525-534). C. elegans (T13F2.2 protein, GenBank gene number Z81122), and S. pombe (CAB10003.1 protein, GenBank gene number Z97185-2)).

Figure 4 is an electrophoresis picture showing genomic Southern hybridization of MIP102. B. campestris genomic DNA was digested with HindIII (lane 1), EcoRI (lane 2) or EcoRV (lane 3) and fractionated through a 10.% agarose gel and transferred to a nylon membrane. This blot was hybridized with the ³² P-labeled 440-bp PstI-HindIII fragment of pGEX-Bc2 (including nucleotides 1-428 of cDNA insertion shown in FIG. 2). The position of the size (bp) marker is shown on the left.

5 is an electrophoretic photograph showing the binding of ToMV MP to the N terminus of MIP102. (A) Affinity-purified GST-MIP102 (lane 1), GST-MIP102dN (lane 2), GST-MIP102dc (lane 3) and GST (lane 4) were analyzed in 10% SDS-polyacrylamide gels And blotted on PVDF membranes. Blots were probed with ² P-labeled PKA-MP after restoration of the protein. (B) The protein profile in the gel used in (A) was revealed by CBB staining. Arrows indicate the location of the recombinant protein. Dashed arrows indicate the occurrence of proteolysis. The position of the molecular weight (kDa) marker is shown on the left.

6 is a photograph showing green fluorescence of plant cells due to receptor proteins for viral cell-to-cell migration. (A) As the viral genome migrates to neighboring cells, green fluorescent proteins are produced within the cells and emit green light (pictured as “multiple cells”). (B) In contrast, if the virus does not perform cell-to-cell migration, fluorescence is produced only within a single cell (pictured as “one cell”).

7 is a graph showing the effect of co-introduction of pART7-Bc2dc-HA and piL.G3 upon cell-to-cell migration. In Figure 7, the vertical axis represents the ratio of cell-to-cell migration expressed by the fluorescence spot ratio (multiple cells / total cell number) while the horizontal axis represents the molar ratio of the plasmid (pART7-Bc2dc-HA / piL.G3). .

8 is a graph showing the effect of co-introduction of pART7-Bc2dc-HA and piL.erG3 (ER-positioned GFP expression plasmid) upon cell-to-cell migration. In Figure 8, the vertical axis represents the ratio of cell-to-cell migration expressed by the fluorescence spot ratio (multiple cells / total cell number), while the horizontal axis represents the molar ratio of the plasmid (pART7-Bc2dc-HA / piL.erG3). .

Figure 9 is a structural diagram showing the expression plasmid pART7-Bc2dc-HA producing the N-terminal binding region of MIP102.

10 is an electrophoresis picture showing the expression of the target protein of the transformed tobacco. From the right in the following order, (A) Coomassie Brilliant Blue (CBB) -stained gel, (B) results of using HA tag antibody and (C) results of using KELP antibody, respectively. The leftmost lane in the resulting lane represents the molecular weight marker and the remaining lanes represent the tobacco strains 101, 102, 105 and 118 transformed in order from the left. The rightmost lane represents the non-transformed tobacco (control).

11 is an electrophoretic photograph showing the results of virus resistance studies due to ToMV particle infection. Opposite end lanes represent molecular weight markers and the remaining lanes are transformed from transformed tobacco strains 101r, 102r, 105r and 118r in order from the left (they were regenerated from transformed tobacco strains 101, 102, 105 and 118) and non-transformation Tobacco (SR1). For each strain the left lane represents the upper leaf (U) while the right lane represents the infected leaf (I). The bands indicated by the arrows correspond to coat proteins.

12 shows an electrophoretic photograph showing the binding of ToMV MP to AtKELP. (A) Affinity-purified GST-AtKELP (lane 1), GST-MIP102 (lane 2) and GST (lane 3) were analyzed on 10% SDS polyacrylamide gels and blotted on PVDF membranes. After restoration of the protein was probed with ³² P-labeled PKA-MP. (B) The protein profile in the gel used in (A) was revealed by CBB staining. Arrows indicate the location of the recombinant protein. The size of GST-MIP102 is larger than GST-AtKELP due to the extra 15 amino acids between GST and MIP102. The position of the molecular weight (kDa) marker is shown on the left.

FIG. 13 is an electrophoretic photograph showing the binding of AtKELP to MPs of various tissues. Equal amounts (1 μg) of affinity-purified GST-MP (ToMV), GST and bovine serum albumininnitrocellulose membranes were immobilized and probed with ³² P-labeled PKA-AtKELP.

According to the present invention there is provided a method for imparting plant virus resistance to a plant. By "implanting plant virus resistance" the disease damage is prevented or minimized even when the plant is infected with the virus. The method of the present invention consists of introducing into the plant cell a polynucleotide encoding a protein capable of binding to the plant virus transfer protein.

Proteins encoded by the polynucleotides used in the methods of the invention bind to the mobile protein (MP) of the infectious plant virus. This interaction blocks the interaction of the infectious viral migration protein with the factors present in the host cell involved in viral migration. Thus, viral migration from cell to cell through plasmidema is blocked. The above-described proteins are expressed in plants and are blocked from plant virus cell-to-cell migration and thus tissue-to-tissue migration (long-range migration). As a result, viral disease damage is prevented or minimized. Thus, the polynucleotides used in the methods of the present invention impart plant virus resistance to plants by expressing proteins capable of binding to plant virus transfer proteins in the plants.

The polynucleotides used in the methods of the invention are screened from the plant cDNA library as polynucleotides encoding proteins capable of binding to the encoded mobile protein (MP) within the plant viral RNA genome. Proteins that can then bind to these mobile proteins are termed mobile protein interacting proteins (MIPs). Moving protein interacting proteins (MIPs) are identified by West Western methods that can search for unknown proteins that can bind known proteins. For example, polynucleotides encoding proteins capable of binding to encoded protein (MP) within the RNA genome of tomato mosaic virus (ToMV) are those of plants such as Nicotiana Tabacum, Arabidopsis Thaliana and Brassica Campestris. Screened from cDNA Library. Such MIPs include, but are not limited to, for example, MIP204, MIP105, and MIP106, derived from Nicotiana Tabacum library, and MIP204 derived from Brassica Campestris library. Of these MIPs, MIP102 shows the highest binding to tomato mosaic virus transfer protein. The nucleotide sequence encoding the same as the amino acid sequence of MIP102 is shown in SEQ ID NO: 2 and SEQ ID NO: 1.

An exemplary mobile protein interacting protein MIP102 binds to a mobile protein through the entire length and N terminal portion of MIP102. Thus in one embodiment the polynucleotide of the method of the invention encodes a protein comprising an amino acid sequence from methionine (Met) at position 1 of SEQ ID NO: 2 in the sequence listing to glycine (Gly) at position 86 . In another embodiment the polynucleotides used in the methods of the present invention encode a protein comprising an amino acid sequence from methionine (Met) at position 1 of SEQ ID NO: 2 in the sequence listing to Valin at position 165 do. The polynucleotides used in the methods of the present invention encode proteins comprising amino acid sequences with one or more amino acid deletions, substitutions and / or additions in which the portion encoded by the polynucleotides has the function of binding to the virus transfer protein. .

In one embodiment the polynucleotides of the invention comprise polynucleotides having the nucleotide sequences of positions 14 to 271 shown in SEQ ID NO: 1 in Sequence Listing. In one embodiment the polynucleotides of the invention comprise polynucleotides having the nucleotide sequences of positions 14 to 508 shown in SEQ ID NO: 1 in Sequence Listing.

Nucleotide sequences disclosed herein as well as fragments and variants of proteins encoded by these sequences are included in the present invention. "Fragment" means a portion of a nucleotide sequence, a portion of an amino acid sequence and a protein encoded by the sequence. Fragments of nucleotide sequences encode protein fragments that retain the functional bioactivity of one or more native proteins. Nucleotide sequences and fragments of the proteins encoded by the sequences disclosed in the methods of the invention can be used as long as they bind to the virus transfer protein.

Variants of a protein encoded by the polynucleotides of the present invention may include one or more amino acid deletions (so-called reductions) or additions at the N- and / or C-terminal ends of the protein; Deletion or addition of amino acids at one or more sites of the protein; Or a protein derived from a native protein by amino acid substitution at one or more sites of the protein. Such variants are produced, for example, by polymorphism or artificial manipulation.

Proteins encoded by the polynucleotides of the present invention are modified by various methods (including amino acid substitutions, deletions, reductions, and insertions). Methods of such manipulations are generally known in the art. For example, amino acid sequence variants of proteins encoded by plant genes capable of modulating stress resistance according to the present invention are produced by mutations in DNA. Methods of mutation and nucleotide sequence changes are well known in the art. See, eg, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Ezymol. 154: 367-382; US Patent No. 4,873,192; See Walker and Gastra, editors, (1983) Techniques in Molecular Biology (MacMillian Publishing Company, New York) and references. Guidance as appropriate amino acid substitutions that do not affect the bioactivity of the protein of interest is described in Dayhoff et al. (1987) found in a model of Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.Washington D.C., incorporated by reference). Conservative substitutions such as exchanging one amino acid for another with similar properties are preferred. Examples of conservative substitutions include hydrophobic amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Tyr, Val); Hydrophilic amino acids (Arg, Asp, Asn, Cys, Glu, Gln, Gly, His, Lys, Ser, Thr); Amino acids with fatty side chains (Gly, Ala, Val, Leu, Ile, Pro); Amino acids with hydroxyl group containing side chains (Ser, Thr, Tyr); Amino acids having a sulfur atom containing side chain (Cys, Met); Amino acids with carboxylic acid and amide containing side chains (Asp, Asn, Glu, Gln); Amino acids with base containing side chains (Arg, Lys, His); Amino acids with aromatic containing side chains (His, Phe, Tyr, Trp), including but not limited to.

Thus, "having one or more deletions, substitutions and / or additions" means that many amino acids substituted, deleted and / or added by polymorphism or artificial manipulation (including the well-known methods described above) are substituted, deleted and / or Means added. In addition, "having one or more deletions, substitutions and / or additions" means that as many amino acids are deleted, added to the above-described amino acid sequence as long as the function of the protein encoded by the polynucleotide of the present invention can be expressed. / Or substituted. It will be apparent to those skilled in the art that the effect of changes in activity, such as amino acid substitutions, deletions and / or additions, will depend on the location, extent, type, etc. of the amino acid being changed. Proteins encoded by the polynucleotides of the present invention have many deletions, substitutions and / or additions within the above-described amino acid sequences, and their numbers are defined as long as the functions of the proteins of the polynucleotides of the present invention are expressed. Satisfy sequence identity.

The polynucleotides used in the methods of the present invention comprise at least 70 amino acid sequences from Met of position 1 of SEQ ID NO: 2 to Gly of position 86 in the sequence listing such that the protein encoded by such polynucleotides binds to the virus transfer protein. Amino acid sequence having at least%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% sequence identity Polynucleotides having a nucleotide sequence encoding a protein having The polynucleotides used in the methods of the present invention comprise at least 70 amino acid sequences from Met of position 1 of SEQ ID NO: 2 to Val of position 165 in the sequence listing such that the protein encoded by such polynucleotides binds to the virus transfer protein. Amino acid sequence having at least%, preferably at least 75%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% sequence identity Polynucleotides having a nucleotide sequence encoding a protein having

The polynucleotides used in the methods of the invention comprise at least 75 amino acid sequences from Met of position 1 of SEQ ID NO: 2 to Gly of position 86 in the sequence listing such that the protein encoded by such polynucleotides binds to the virus transfer protein. Amino acid sequence having at least%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% sequence identity Polynucleotide having a nucleotide sequence encoding a protein having (preferably the nucleotide sequence from position 14 of position 14 of SEQ ID NO: 1 to position A of position 271). The polynucleotides used in the methods of the invention comprise at least 75 amino acid sequences from Met of position 1 of SEQ ID NO: 2 to Val of position 165 in the sequence listing so that the protein encoded by such polynucleotides binds to the virus transfer protein. Amino acid sequence having at least%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95% and most preferably at least 99% sequence identity Polynucleotide having a nucleotide sequence (preferably a nucleotide sequence from position A of position 14 of SEQ ID NO: 1 to position C of position 508 C) encoding a protein having

The polynucleotides of the present invention may contain a nucleotide sequence (ie, a non-translated region) that is additional to the outside (ie, 5 'or 3' end) of the nucleotide sequence encoding a protein comprising the amino acid sequence of the above-described region. Include. Preferably the polynucleotide used in the method of the present invention consists of the full length sequence of positions 1 to 913 of SEQ ID NO: 1. The polynucleotides of the present invention include all degenerate isomers of SEQ ID NO: 1. As used herein, the term "degenerate isomer" refers to DNA capable of encoding the same polypeptide that differs only by the degenerate codon (s). For example, DNA is called a degenerate isomer in which a codon corresponding to an amino acid associated with the DNA having the nucleotide sequence of SEQ ID NO: 1 is replaced by its degenerate codon (ie, codon AAT).

As used herein, a “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence is a subset or whole of a specialized sequence: eg a segment of a full-length cDNA or polynucleotide sequence or a complete cDNA or polynucleotide sequence.

As used herein, a “comparison window” means is compared to a reference sequence and its portion within the comparison window added or compared to the reference sequence (not consisting of additions or deletions) for optimal alignment of the two sequences. Reference to contiguous and specified segments of the polynucleotide sequence consisting of deletions (ie, gaps). In general, the comparison window is at least 20 contiguous nucleotides in length, optionally 30, 40, 50, 100 or longer. Those skilled in the art will generally introduce gap penalties and subtract from the number of matches to avoid high similarity to the reference sequence due to the inclusion of gaps in the polynucleotide sequence.

Methods for aligning sequences for comparison are well known in the art. A preferred method for determining the best overall match between a reference sequence (a sequence of the invention) and an experimental sequence is homology analysis using BLAST (Altshul et al., 1997, Nucleic Acids Res., 25, 3389-3402). In sequence alignment both the reference sequence and the experimental sequence are DNA sequences. RNA sequences are compared by converting U's to T's. The result of this global sequence alignment is percent identity. DNA sequence alignments are performed using BLAST's default parameters to calculate percent identity.

As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences is a reference to residues in the same two sequences when aligned in maximal correspondence to a specified comparison window. It includes. When a percentage of sequence identity is used as a reference to a protein, the amino acid residues are replaced by other amino acid residues with similar chemical properties (ie, charge or hydrophobicity) and are therefore not identical by conservative amino acid substitutions that do not change the functional properties of the molecule. Residue positions are often different. If the sequences differ in conservative substitutions, the percent sequence identity is adjusted in a way to correct the conservative nature of the substitution. Sequences changed by such conservative substitutions have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. In general this increases the percentage of sequence identity, including scoring partially conservative substitutions rather than overall mismatches. Thus, for example, if the same amino acid is given a score of 1 and a non-conservative substitution is given a score of 0, conservative substitutions are given a score between 0 and 1. Scoring of conservative substitutions is performed and calculated in program PC / GENE (Intelligenetics, Mountain View, California).

As used herein, “percentage of sequence identity” means that a portion of the polynucleotide sequence in the comparison window is added or deleted (ie, gaps) compared to the reference sequence (not consisting of additions or deletions) for optimal alignment of the two sequences. Means the value determined by comparing two optimally aligned sequences in a comparison window consisting of Percentage measures the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, divides the number of matched positions by the total number of positions in the comparison window, and percentage of sequence identity It is calculated by multiplying the result by 100 to yield.

The term "substantial identity" of a polynucleotide sequence is at least 80%, preferably at least 90%, most preferably at least 95% of the sequence compared to the reference sequence using one of the alignment programs described using standard parameters. It consists of a sequence with identity. Those skilled in the art will recognize that these values are appropriately adjusted to determine the corresponding identity to the protein encoded by the two nucleotide sequences by taking into account codon degradation, amino acid similarity, and reading frame position. Substantial identity of amino acid sequences for these purposes generally means sequence identity of at least 70% or more, preferably at least 75%, 80%, 90% or more, most preferably at least 95% or more.

The term "substantial identity" within the context of a peptide refers to a sequence having at least 70%, preferably at least 80%, more preferably 90% or 95% or more sequence identity to a reference sequence in the peptide's specified comparison window. It consists of. Preferably the optimal alignment is Needleman et al., J. Mol. Biol. 48: 443 (1970) using the homology alignment algorithm. Thus, one peptide is substantially the same as the second peptide, for example if the two peptides differ only by conservative substitutions. “Substantially similar” peptides share a sequence as noted above, except that non-identical residue positions are changed only by conservative amino acid exchange. The GENETYX program is used to compare peptide identity. In this case, the program's default parameters are used.

Polynucleotide fragments encoding biologically active protein portions encoded by polynucleotides used in the methods of the invention may comprise at least 15, 25, 30, 50, 100, 125, 150, 175, 200, or 250 contiguous amino acids. Or the total number of amino acids of the full length protein used in the methods of the invention (ie, 243 amino acids of SEQ ID NO: 2). In general, fragments of nucleotide sequences capable of conferring viral resistance to plants used as hybridization probes of PCR primers may identify bioactive parts of proteins expressed by polynucleotides capable of conferring plant viral resistance. Code it.

Typical mobile proteins that interact with protein MIP102 are derived from Brassica campestris. Polynucleotides used in the methods of the invention also include polynucleotides encoding mobile proteins that interact with proteins derived from plants other than Brassica campestris. Such polynucleotides can be subjected to PCR using primers designed based on, for example, all or part of a nucleotide disclosed as a template and genomic DNA of a selected plant, and then using the resulting amplified DNA fragment as a probe and using genomic DNA or Isolation by screening cDNA libraries. In this way methods such as PCR, hybridization, and the like are used to identify sequences based on sequence identity to the sequences disclosed herein. Sequences separated based on their sequence identity to all or fragments thereof disclosed herein are included in the present invention.

All or part of the known nucleotide sequences in the hybridization method are optionally hybridized to cloned genomic DNA fragments or other corresponding nucleotide sequences present in a group of cDNA fragments (ie, genomic libraries or cDNA libraries) derived from selected organisms. It can be used as a probe to size. These hybridization probes are genomic DNA fragments, cDNA fragments, RAN fragments or other oligonucleotides and are labeled with detectable groups such as ³² P or other detectable markers. The preparation of probes for hybridization and structure of cDNA libraries and genomic libraries is generally known in the art and disclosed in Sambrook et al. (1989). Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

For example, all or one or more portions of the nucleotide sequence of a plant gene capable of conferring plant virus resistance disclosed herein are specifically hybridized to sequences of plant genes and messenger RNAs thereof capable of conferring viral resistance to the plant. It is used as an easy probe. To achieve specific hybridization under a variety of conditions, such probes are unique among sequences of plant genes capable of imparting viral resistance to plants, preferably at least 10 nucleotides in length, most preferably 20 nucleotides in length. It includes the above sequence. Such probes are used to PCR amplify the sequences of plant genes capable of conferring viral resistance to plants from selected organisms. PCR amplification methods are well known in the art (PCR Technology: Principles and Applications for DAN Amplification, HA Erlich ed., Freeman Press, New York, NY (1992); PCR Protocols: A Guide to Methods and Applications, Innis, Gelfland , Snisky, and White, eds., Academic Press, San Diego, CA (1990); Mattila et al., (1991) Nucleic Acids Res. 19: 4967; Eckert, KA and Kunkel, TA (1991) PCR Methods and Applications 1: 17; PCR, McPherson, Quirkes, and Taylor, IRL Press, Oxford). This technique is used in diagnostic assays to separate additional coding sequences from a desired organism or to measure the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (plaques or colonies: ie Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed., Cold Spring Harbor Laboratory Press, Plainview, New York)).

Such sequence hybridization is performed under stringent conditions. The terms “stringent condition” or “stringent hybridization condition” refer to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than other sequences (ie, at least twice as much as background). Include. Stringent conditions are sequence-dependent and will vary in other situations. By controlling the stringency of hybridization and / or wash conditions, a target sequence that is 100% complementary to the probe will be identified. Stringency conditions can also be adjusted to allow some mismatches in the sequence to detect a lower degree of similarity. In general, the probe is about 1000 nucleotides or less in length, preferably 500 nucleotides or less.

In general, stringent conditions are at a salt concentration of about 1.5 M Na ions or less, typically about 0.01 to 1.0 M Na ion concentration (or other salts), and for short probes (ie, 10 to 50 nucleotides) at least about At least 30 ° C. (pH 7.0-8.0), for long probes (ie at least 50 nucleotides) will be at least about 60 ° C. or more. Stringent conditions are also achieved with the addition of destabilizing agents such as formamide. Typical low stringent conditions are at 42 ° C. in a solution containing 50% formamide, 4.4 × SSC, 20 mM phosphate buffer (pH 6.8), 1 × Denhardt's solution, 0.2% SDS and denatured salmon sperm DNA (0.1 mg / ml). Washes at room temperature in 2 × SSCs containing hybridization and 0.1% SDS and final wash at 42 ° C. in 0.2 × SSCs containing 0.1% SDS.

Specificity is generally the function of the post-hybridization wash, an important factor in ionic strength, and the temperature of the final wash solution. For DNA-DNA hybrids, T _m is Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984) can be estimated from the equation: T _m = 81.5 ° C + 16.6 (log M) + 0.41 (% GC)-061 (% form)-500 / L; M is the mole of monovalent cation,% GC is the percentage of guanine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in the base pair. T _m is the temperature at which 50% of the complementary target sequences hybridize to a perfectly matched probe (under defined ionic strength and pH). T _m decreases by about 1 ° C. for each 1% mismatch; Thus T _m , hybridization and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if a sequence with> 90% identity is searched for, T _m may be reduced to 10 ° C. In general, the Sturgent conditions are chosen to be about 5 ° C. below the thermal melting point (T _m ) for the specific sequence and its complementarity at defined ionic strength and pH. However severe stringent conditions may utilize hybridization and / or washing at temperatures below 1, 2, 3 or 4 ° C. below the thermal melting point (T _m ); Normal stringent conditions may utilize hybridization and / or washing at temperatures below 6, 7, 8, 9 or 10 ° C. below the thermal melting point (T _m ); Low stringent conditions may utilize hybridization and / or washing at temperatures below 11, 12, 13, 14, 15 or 20 ° C. below the thermal melting point (T _m ). Using equations, hybridization and wash compositions, and the desired T _m , those skilled in the art will understand that the various stringencies of hybridization and wash solutions are uniquely described. If the desired degree of mismatching results in a lower T _m than 45 ° C. (aqueous solution) or 32 ° C. (formamide solution), it is desirable to increase the SSC concentration so that higher temperatures are used. Extensive guidance on hybridization of nucleic acids can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Moleculuar Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); And Ausubel, et al., Eds. (1995), Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plaineview, New York) (these are incorporated by reference).

The base sequence of the obtained gene is determined by nucleotide sequence analysis known in the art or a commonly available automated sequencer.

Polynucleotides of the invention are generally obtained according to the methods disclosed herein or by chemical synthesis based on the sequences disclosed herein. For example, the polynucleotides of the present invention are synthesized using a polynucleotide synthesizer (Applied BioSystem (at present, Perkin Elmer)) according to the manufacturer's instructions.

Polypeptides produced according to procedures such as genetic engineering techniques or chemically synthesized techniques, as described above, are identified as having the desired activity, i.e., to impart viral resistance as follows. Expression vectors containing polynucleotides are generated. Such expression vectors are expressed in suitable cells that produce proteins. Substantially the same procedure as described in Example 1 below is used to determine whether the protein binds to the desired plant virus transfer protein (MP). Substantially the same procedure as described in Example 4 below confirms that when the protein is introduced into the plant cell with the virus, the protein inhibits cell-to-cell migration of the virus where receptor genes such as green fluorescent genes are used as indicators It is used to

The polynucleotides used in the methods of the invention confer plant viral resistance to plants. Plant viruses are not always specific. A mobile protein interacting protein capable of binding to a specific virus's mobile protein binds to another viral's mobile protein. Also, for example, MIP102 and AtKELP (proteins having about 75% amino acid sequence identity over the entire length of MIP102), which are common proteins capable of binding to the tomato mosaic virus mobile protein, can also be found in the Brassicaceae mosaic virus ( crucifer tobamovirus ) and Combine with cucumber mosaic virus.

In the examples presented herein, viral resistance to tomato mosaic virus (ToMV) is shown for purposes of illustration. The method of the present invention also confers resistance to other plant viruses on the plant. Examples of plant viruses that have been tested for resistance include Tobamovirus , Tobravirus , Dianthovirus , Alfamovirus , Bromovirus , and Cuckoo. Cucumovirus , Comovirus , Nepovirus , Caulimovirus , Geminivirus , Potivirus and Tospovirus Including but not limited to viruses. The polynucleotides used in the method of the present invention are obtained by identifying plant virus transfer proteins of plants to which resistance is imparted, and screening for proteins capable of binding to the transfer proteins identified by the West Western screening method as described in Example 1 do.

The polynucleotides of the present invention are linked to appropriate plant expression vectors using methods well known to those skilled in the art and introduced into the plant cells by known recombinant techniques. The introduced gene is integrated into the DNA in the plant cell. DNA in plant cells includes chromosomes as well as DNA contained in various organelles (ie mitochondria, chloroplasts, etc.) within plant cells.

As used herein, a “plant expression vector” refers to a nucleic acid sequence in which various regulatory sequences, such as promoters for controlling gene expression of the invention, are operatively linked in a host plant cell. As used herein, the term “control sequence” refers to a DNA sequence with a functional promoter and any related transcriptional elements (ie, enhancers, CCAAT boxes, TATA boxes, and SPI sites). As used herein, the term “practically linked” refers to the polynucleotide being linked to regulatory elements that regulate gene expression such as a promoter or enhancer such that the gene can be expressed. Plant expression vectors preferably include plant gene promoters, terminators, drug-resistant genes and enhancers. It is well known to those skilled in the art that the type of expression vector used and the type of regulatory element depend on the host cell. The plant expression vector used in the present invention has a T-DNA region. The T-DNA region can enhance the efficiency of transduction, especially when Agrobacterium is used to transform plants.

The term "plant gene promoter" as used herein refers to a promoter expressed in a plant. Plant promoter fragments may be used that will direct the expression of the polynucleotides of the invention in all tissues of the regenerated plant. Examples of promoters for structural expression include promoters of the nopaline synthase gene (Langride, 1985, Plant Cell Rep. 4, 355), promoters for generating cauliflower mosaic virus 19S-RAN (Guilley, 1982, Cell 30, 763). ), A promoter for generating cauliflower mosaic virus 35S-RNA (Odell, 1985, Nature 313, 810), rice actin promoter (Zhang, 1991, Plant Cell 3, 1155), corn ubiquitin promoter (Cornejo 1993, Plant Mol. Biol. 23, 567) and REXφ promoter (Mitsuhara, 1996, Plant Cell Physiol. 37, 49).

In addition, plant promoters may direct the expression of the polynucleotides of the present invention in a particular plant or will be under more precise environmental or developmental control. Such promoters are referred to herein as "inducible" promoters. Examples of inducible promoters include promoters that are inducible by environmental conditions such as infection or pathogen invasion, plant wounds, light, low temperature, high temperature, drying, UV irradiation, injection of certain compounds, and the like. Examples of such promoters include promoters of genes encoding ribulose-1,5-diphosphate carboxylase small subunits induced by light irradiation (Fluhr, 1986, Proc. Natl. Acad. Sci. USA 83, 2358), promoters of rice chitinase genes expressed by infection or invasion of molds, bacteria or viruses (Xu, 1996, Plant Mol. Biol. 30, 387). , Promoter of tobacco PR protein gene (Ohsima, 1990, Plant Cell 2, 95), promoter of rice lip19 gene inducible by low temperature (Aguan, 1993, Mol. Gen. Genet. 240, 1), Promoter of rice hsp72 and hsp80 genes induced by high temperature (Van Breusegem, 1994, Planta 193, 57), promoters of the rab16 gene of Arabidopsis thaliana inducible by drying (Nundy, 1990, Proc. Natl. Acad. Sci. USA 87, 1406) and Corn Inducible by Ultraviolet Irradiation And a promoter of alcohol dehydrogenase gene (Schulze-Lefert, 1989, EMBO J. 8, 651). The promoter of the rab16 gene is inducible by spraying the plant hormone abstic acid.

A “terminator” is a sequence located downstream of the region that encodes a portion of a gene and associated with terminating transcription and adding poly A sequence when DNA is transcribed into mRNA. Terminators are known to contribute to mRNA stability and affect gene expression levels. Examples of such terminators include, but are not limited to, CaMV35S terminators and ternos of nopaline synthase.

The "drug-resistant gene" is preferably one that facilitates the screening of transformed plants. Preferably, the neomycin phosphatase II (NPTII) gene which confers kanamycin resistance, the hygromycin phosphatase gene which confers hygromycin resistance, etc. are used. The present invention is not limited thereto.

"Enhancers" are used to enhance the expression efficiency of the gene of interest. As an enhancer, an enhancer region containing an upstream sequence in the CaMV35S promoter is preferred. Multiple enhancers are used for each plant expression vector.

If polypeptide expression is desired, it is generally preferred to include a polyadenylation region at the 3 'end of the polynucleotide coding region. The polyadenylation zone can be derived from natural genes, various other plant genes or T-DNA. The 3 'terminal sequence to be added may be derived from, for example, a nopaline synthase or octopine synthase gene or other plant gene or less preferably another eukaryotic gene.

As described above, plant expression vectors are prepared using genetic recombination techniques well known to those skilled in the art. When constructing a plant expression vector, pBI vectors, pUC vectors, pART vectors and the like are preferably used. The present invention is not limited thereto.

Plant material for the introduction of DNA may be appropriately selected from leaves, stems, roots, tubers, protoplasts, callus, pollen, embryos, early chute, etc., depending on the introduction method. "Plant cells" can be any plant. Examples of “plant cells” include cells of tissues in plant organs such as leaves and roots; Callus; And cultured cell suspensions. The plant cell may be a cultured cell, cultured tissue, cultured organ or any form of plant. Preferably the plant cell is a culture cell, culture tissue or culture organ. More preferably the plant cells are cultured cells.

Plant culture cells into which DNA is introduced are generally protoplasts. DNA is introduced into plant culture cells by physical / chemical methods such as electroporation methods, polyethylene glycol methods, and the like. Plant tissues into which DNA is introduced are leaves, stems, roots, tubers, callus, pollen, embryos, early chutes, preferably leaves, stems and callus. DNA is introduced into plant tissue by physical or chemical methods such as biological or particle gun methods using viruses or Agrobacterium. For example, a method using Agrobacterium is disclosed in Nagel et al. (Microbiol. Lett., 67, 325 (1990)). In this method the plant expression vector is first used to transform Agrobacterium and then the transformed Agrobacterium is introduced into plant tissue by well known methods such as the leaf disk method. For example, particle counting methods are described in Klein et al. (1987), Nature 327: 70-73; And Christon, P. Plant J. (1992) 2, 275-281. The protocol of transformation and introduction of the nucleotide sequence into the plant depends on the type of target plant or plant cell (ie, monocotyledonous or dicotyledonous plant) to be transformed. Examples of suitable methods for introducing nucleotide sequences into plant cells to insert sequences into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electrophoresis (Riggs et al. 1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606), Agrobacterium mediated transformation (Townsend et al., US Pat. No. 5,563,055), direct gene transfection (Paszkowski et al. 1984) EMBO J. 3: 2717-2722) and ballistic particle acceleration (ie, Sanford et al., US Pat. No. 4,945,050; Tomesra, US Pat. No. 5,879,918; Tomes et al., US Pat. No. 5,886,244; Bideny et al., US Pat. No. 5,932,783; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment", Plant Cell, Tissue, and Organ Culture: Fundamental Methods, Gamborg and Phillips ed., (Springer Verlag, Berlin) and McCabe et al. (1988) Biotechnology 6: 923-926). See also the following references: Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1988) Theor. Appl. Genet. 96: 319-324 (soy); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (corn); Klein et al. (1988) Biotechnology 6: 559-563 (corn); Tomes, U.S. Patent 5,240,855; Buising et al., US Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment", Plant Cell, Tissue, and Organ Culture: Fundamental Methods, Gamborg ed. (Springer-Verlag, Berlin) (corn); Klein et al. (1988) Plant Physiol. 91: 440-444 (corn); Fromm et al. (1990) Biotechnology 8: 833-839 (corn); Hooykaas-Van Slongteren et al. (1984) Nature (London) 311: 763-764; Bowen et al., US Pat. No. 5,736,369 (crop species); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Lilies); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues, Chapmann et al. eds. (Longman, New York) pp. 197-209 (flower pots); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker mediated transformation); D 'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (corn (via Agrobacterium tumefaciens)), all of which are incorporated by reference. These methods are well known in the art. Suitable methods for the plant to be transformed may be appropriately selected.

Cells into which plant expression vectors have been introduced are selected for drug resistance, such as kanamycin resistance. Transformed cells can be regenerated by methods commonly used. For example, McCormick et al. (1986) Plant Cell Reports 5: 81-84.

Plant cells into which the polynucleotides of the present invention are introduced can be regenerated into plants by culturing the plant cells in regeneration medium, hormone-free MS medium. Plants with young roots can be grown into plants by transferring them to soil and growing them. Regeneration methods vary depending on the type of plant cell. Regeneration methods for various plants are described: rice (Fujimura, 1995, Plant Tissue Culture Lett. 2, 74); Corn (Shillito, 1989, Bio / Technol. 7, 581; Gorden-Kamm, 1990, Plant Cell 2, 603); Potatoes (Visser, 1989, Theor. Appl. Genet. 78, 594); And tobacco (Nagata, 1971, Planta 99, 12).

Expression of the introduced genes of the present invention in regenerated plants can be confirmed by methods well known to those skilled in the art. This confirmation is for example performed using Northern blotting analysis. In particular, total RNA is extracted from the plant leaves and electrophoresed on denatured agarose and blotted to a suitable membrane. This blot is hybridized with labeled RNA probes complementary to the introduced gene moiety to detect mRNA of the genes of the invention. These plants are then grown and identified with the same transformed strain or other strains and the resulting hybrid with the desired phenotypic properties. Two or more generations are grown to confirm that the subject phenotypic properties remain stable, inherited and seed harvested to achieve the desired phenotype or other properties.

Plants that can be produced by the methods of the invention include any plant into which a gene can be introduced. As used herein, the term "plant" includes whole plants, plant organs (ie, leaves, stems, roots, etc.), seeds, plant propagators (ie, pollen), and plant cells and their progeny. Plant cells as used herein include, but are not limited to, seeds, culture suspensions, embryos, meristem parts, callus tissues, leaves, roots, chutes, spores, spores, pollen and vesicles. The term "plant" includes monocots and dicotyledons. Such plants include any useful plant, particularly crop plants, vegetable plants and flowering plants of garden species. The most preferred plant to which the present invention is applied is tobacco.

Examples of plant types that can be used in the present invention include Solanaceae (Solanaceae), hwabongwa (Poaeae), mustard (Brassicaceae), Rosaceae (Rosaceae), leguminous (Leguminosae), pumpkin and (Cucurbitaceae), Lamiaceae (Lamiaceae), lily and (Liliaceae), comprises chenopodiaceae (chenopodiaceae), and the mountain-shaped (Umbelliferae) plant.

Examples of the Solanaceae plant includes tiahna in Nicosia (Nicotiana), Solar numsok (Solanum), Datu rasok (Datura), Fernand Rico in Sion (Lycopersion) and Petunia spp. Specific examples include tobacco, eggplant, potatoes, tomatoes, chili peppers, and petunias.

Examples of flowering plants and plants include rice, barley, rye, sugar cane, Echinochloa and corn plants. Specific examples include rice, barley, rye, peels, sugar cane and corn.

Examples of mustard plants include genus Radix , Brassica, Arabidopsis, Wasabia and Capsella . Specific examples include Japanese white radish, rape seed, Arabidopsis thaliana, Japanese horseradish and wasabi.

Examples of the Rosaceae plant include Orunus , Cactaceae , Pynus , Fragaria and Rosaceae . Special examples include plums, peaches, apples, pears, Danish strawberries and roses.

Legumes Examples include legumes, Vigna , kidney beans, peas, rakes, peanuts , shamrocks, alfalfa, and canis plants. Specific examples include soybeans, adzuki beans , kidney beans, peas, green beans, peanuts, clover and bur clover .

Zucchini plants include Luffa , Cucurbita and Lilium plants. Specific examples include gourds, pumpkins, cucumbers and melons.

Lamiaceae and plants include Lavandula, Mint, and Chaz. Specific examples include lavender, peppermint and beefsteak plants.

Lilium plants include the genus Garlic, Lilium and Tulip. Specific examples include onions, garlic, lilies, and tulips.

Chenopodiaceae plants include spinach in (Spinacia) plants. A special example is spinach.

The cultivars include the genus Pinus , Carrot, Cryptotaenia and Celery. Specific examples include Japanese udo, carrots, honewort and celery.

Viral resistance conferred by the method of the present invention is inherited in the next generation of plants as well as the initial generation of transformed plants. Viral resistance conferred by the peptides of the present invention is manifested in the initial generation of the transformed plant and the next generation of the plant, its breeders (ie pollen) and seeds generated from the breeder. The heritability to the next generation of polynucleotides used in the methods of the present invention is confirmed by Southern blot using the sequences of the polynucleotides disclosed herein as probes.

The names used hereafter and the experimental procedures described thereafter include procedures well known and commonly used in the art. Standard techniques are used for recombinant methods, polynucleotide synthesis, cell culture, transformation and plant regeneration. Techniques and procedures are generally performed according to conventional methods and various general references in the art (generally Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY).

The invention will now be described by way of examples. The present invention is not limited thereto. Materials, reagents, and the like used in the examples are conventionally used unless otherwise stated.

Example 1 Far-Western (“West Western”) Screening of Mobile Protein Interacting Protein (MIP)

To identify plant proteins that bind ToMV MP, we rescued N. tabacum and B. campestris expressing cDNA libraries of λGEX5. The structure of the cDNA library is as follows. N. tabacum cv. Structures of leaves from Samsun NN, stigma from B. campestris , and buds from A. thaliana eco-type Colombia were RNA isolated with the QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech), resulting in a TimeSaver cDNA Synthesis Kit (Amersham Pharmacia Biotech). CDNA was synthesized. λ phage vector λ GEX5 (Fukunaga et al., (1997) EMBO J. 16, 1921-1933; these vectors have IPTG inducible promoter and cDNA-cloning site downstream of the GST reading frame) into the structure of the cDNA expression library. Was used. Phosphorylated oligonucleotide adapters (5'-AGGTGCTGG-3 ', 5'-CCAGCACCTGCA-3') were annealed and ligated to the cDNA to make ends compatible with the SfiI-cut vector. The cDNA was ligated with the vector arm and packaged in vitor. Phage libraries were amplified in E. coli strain BB4.

Far-western screening of MP interacting protein (MIP) was performed by GST-fusion ToMV MP phosphorylated with [γ- ³² P] ATP for probing with each GST-fusion cDNA product adhered on the membrane (GST-PKA-MP ) Was performed based on protein-protein binding between

GST-fusion ToMV MP (GST-PKA-MP) phosphorylated with [γ- ³² P] ATP was prepared as follows.

First ToMV MP expression plasmids were prepared as follows. Plasmid pGEX-30K for expression of glutathione-S-transferase (GST) -fusion ToMV MP (GST-MP) has been described previously (Matsushita et al., (2000) J. Gen. Virol 81, 2095-2102). Synthetic oligonucleotides 30K-PKA1 (5'-AATTCGTCGTGCATCTGTTGC-3 ') and 30K-PKA2 (5'-) encoding a five amino acid RRASV to add consensus phosphorylation sequence for protein kinase A between GST and MP AAT TGCAACAGATGCACGACG-3 ') was annealed and inserted into the EcoRI site of pGEX-30K in the proper orientation to produce pGEX-PKA-30K. This plasmid was used for the production of recombinant protein GST-PKA-MP. The 1.1-kb EcoRI-NotI site insertion of pGEX-PKA-30K was located between the EcoRI and NotI sites of the pGEX-6P-2 vector to rescue pGEX-6P2-PKA-30K. This plasmid, which can be cleaved by PreScission Protease (Amersham Pharmacia Biotech) to remove GST and prepare recombinant protein PKA-MP, was used to generate GST-P-PKA MP.

The structures of the pGEX-30KdA and pGEX-30KdSX plasmids have been described above (Matsushita et al., homology). Recombinant protein GST-MPdA encoded by pGEX-30KdA had a deletion of 9 C-terminal amino acids replaced by 27 artificial residues (QVALFGEMCAEPLFVYFSKYIQICIRS) derived from the vector. Another recombinant protein GST-MPdSX encoded by pGEX-30KdSX had a C-terminal 31 amino acid deletion replaced by seven residues (LERPHRD) derived from the vector.

Generation and purification of the recombinant protein was performed as follows. Recombinant GST-fusion protein was generated in E. coli XL10-Gold (Stratagene) with the appropriate plasmid and purified by using Glutathione Sepharose beads (Amersham Pharmacia Biotech) as described above. Anti-GST antibody (goat) purchased from Amersham Pharmacia Biotech was used to identify the fusion protein by Western blotting analysis. Purified protein was stored in NETN-D buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM DTT).

³² P-labeled protein probes were prepared as follows. Glutathione Sepharose beads conjugated with about 1 μg of recombinant GST-fusion protein catalyzed 3.7 MBq of [λ- ³² P] ATP (168 TBq / nmol) and 10 units of protein kinase A (New England BioLabs). It was suspended in 200 μl of kinase buffer (50 mM Tris-HCl, 10 mM MgCl ₂ , pH 8.5) containing subunits. This reaction was performed for 30 minutes on a rotator at 30 ° C. and terminated by washing four times with 1 ml of 50 mM Tris-HCl (pH 8.0) buffer. Phosphorylated protein was eluted from beads with 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM glutathione. Phosphorylated GST-fusion protein at the labeled site was digested with PreScission Protease to remove the GST domain before use as a probe for binding experiments. Specific radioactivity of the probe was about 1 × 10 7 cmp / μg protein.

Far-Western screening of the cDNA library was performed as follows. E. coli strain BB was used for infection with phage libraries. The bacteria were incubated at 42 ° C. for 3 hours to obtain plaques with a density of 100-200 / cm 2. The bacterial plates were covered with a nitrocellulose filter moistened with 10 mM IPTG and incubated at 37 ° C. for 3.5 hours. The filter was washed and the blocking solution, Block Ace (Dainippon-) for 1 hour at 4 ° C. before incubation at 4 ° C. for 16 hours with ³² P-labeled GST-PKA-MP (2 × 10 ⁵ cpm / ml). Incubated in Pharm Co.). The filters were then washed four times for 5 minutes each in PBS (137 mM NaCl, 2.68 mM KCl, 10.14 mM Na ₂ HPO ₄ , 1.76 mM KH ₂ PO ₄ , pH 7.4) with 0.2% Triton X-100 and BAS1500 system ( Radiographs were taken on a Fuji Phto Film.

Some positive clones such as MIP204 from N. tabacum and MIP102, MIP105 and MIP106 from B. campestris were isolated. Of these positive clones, MIP102 showed the highest binding activity and thus was selected for further analysis.

Phage DNA isolated from the MIP102 clone was used to restructure the GST-fusion MIP102 producing expression plasmid pGEX-Bc2 used for protein-binding assays (FIG. 1).

Protein-protein interactions were measured by binding assays between ³² P-labeled protein probes and target proteins fixed on the membrane. The target protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore). After washing with buffer A (50 mM Tris-HCl, 20% 2-propanol, pH 8.0) and buffer B (50 mM Tris-HCl, 5 mM β-mercaptoethanol, pH 8.0) for 1 hour at room temperature, the membrane was then Incubated at room temperature for 1 hour in Buffer B containing 6M guanidine-HCl for denaturation. The membranes were washed for 5 minutes for rerenation and incubated overnight at 4 ° C. in Buffer B containing 0.04% Tween 40. For non-denatured systems the protein was immobilized directly onto the nitrocellulose membrane using a slot-blotter. Membranes were treated with Block Ace for 30 minutes at 4 ° C and ³² P-labeled protein probes in Block Ace with DNase I (18 μg / ml) and Rnase A (60 μg / ml) for 4 hours at 4 ° C. Incubated. The filter membranes were washed four times for 5 minutes each with PBS containing 0.2% Triton X-100 and radiographed. The results are shown in FIG.

48-kDa protein was observed when induced with IPTG on Coomassie Brilliant Blue (CBB) -stained gel (FIG. 1A lane 2) whereas no signal was observed in non-induced lanes (FIG. 1B, lane 1).

GST-MIP102 and GST were subsequently purified using Glutathione Sepharose beads (FIG. 1C) and protein-bound assays were used with the same probes (FIG. 1D). Purified GST-MIP102 showed a positive binding signal (FIG. 1D, lane 1), whereas purified GST did not (FIG. 1D, lane 2). This result indicated that the MIP102 clone was isolated by specific interaction with MP; It was not probed by the GST-GST interaction.

Example 2 Analysis of cDNA and Genomic DNA of MIP102

The nucleotide sequence of MIP102 cDNA and its putative amino acid sequence were determined as follows.

Phage DNA was prepared from the plate lysate of each clone by using the QIAGEN λ kit. Phage DNA was digested with NotI to obtain a DNA fragment corresponding to the pGEX-PUC-3T vector with cDNA insertion for further analysis of cDNA. These DNA fragments were self-ligated to transform E. coli with recovered plasmid clones. Plasmid pGEX-Bc2 recovered from phage clone MIP102 was used to generate the GST-fusion protein GST-MIP102.

Nucleotide sequences were determined using the following primers: pGEX1 primer (5'-GCAAGCCACGTTTGGTGGTG-3 '), pGEX5 primer (5'-ATTTCCCCGAAAAGTGCCAC-3'), Bc2F03 primer (5'-GAGCTTCCTTCTTCTAAAGG-3 '), Bc2R02 primer ( 5'-GCTTCGA TAGCTGGAATATT-3 ').

The nucleotide sequence of MIP102 cDNA is shown in FIG. 2. Homology screening using BLAST showed that the putative protein was similar to the transcriptional coactivator of A. thaliana (AtKELP; Cormack et al., (1998) Plant J. 14, 685-692) (FIG. 3). . The GENETYX program was used to assess the percentage of peptide identification. MIP102 had 36% identity to A. thaliana 's KIWI (Cormack et al., (1998) Plant J. 14, 685-692) (FIG. 3). Also related proteins human (PC4 / p15, 36% identity) (Ge and Roeder, (1994) Cell 78, 513-523; Kretzshmar et al., Homolog) and S. pombe (protein ID CAB10003 in gene bank Z97185). Protein with 1; 19% identity), as well as other toxonomically distinct organisms. Based on the N-terminal homology with AtKELP, the first ATG codon was estimated to be the start codon of MIP102. The open reading frame (ORF) encoded a 165 amino acid polypeptide with a calculated molecular weight of 19,227 Da and pi 4.8. Highly conserved regions related to the single-helix DNA-binding activity of PC4 / p15 were identified (Kretzschmar et al., Homology) (FIG. 3). The central zone of MIP102 is rich in glutamine and glutamic acid (amino acid positions 66-81). Homopolymeric glutamine stretch has been reported to increase transcription factor potential (Gerber et al., (1994) Science 263, 808-811; Schwechheimer et al., (1998) Plant Mol. Biol) 36, 195-204).

Genomic DNA fragments were amplified by PCR and their nucleotide sequences measured to analyze the gene organization of MIP102. Genomic DNA was isolated from the leaves of B. campestris by the phenol / SDS method (Kingston, (1997) Phenol / SDS method for plant RNA preparation. In "Current Protocols In Molecular Biology" (VB Chanda Eds) Vol. 1, pp. John Wiley & Sons, Inc., New York) .Genomic Southern Hybridization was published by Sambrook et al., (1989) "Molecular Cloning A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring. It was performed as described in Harbor, New York. DAN specimens (10 μg) were digested with appropriate restriction enzymes and fractionated through 1.0% agarose gels and transferred onto positively charged nylon membranes (Millipore). The blot was 42 ° C. for 16 h in a solution containing 50% formaldehyde, 4.4 × SSC, 20 mM sodium phosphate buffer (pH 6.8), 1 × Denhardt's solution, 0.2% SDS and denatured salmon sperm DNA (0.1 mg / ml). Hybridized to ³² P-labeled probes. Membranes were washed several times in 2x SSCs containing 0.1% SDS at room temperature before final washing at 42 ° C for 30 minutes in 0.2x SSCs containing 0.1% SDS.

Genomic DNA fragments corresponding to MIP102 cDNA were amplified by PCR using two sets of primers: Bc2F04 (5'-GAAAACCCTAAAGATGGAG-3 ') and Bc2R02 (described above); The Bc2F05 (5'-AATAAGCTTAACAGACGAAC-3 ') and Bc2R07 (5'-GATTTTAAAAGATCATTTTTGTCAT-3') PCR products were cloned into the TA cloning vector pCR2.1 (Invitrogen) and the nucleotide sequence was transformed into three independent clones using vector primers. Measured: Forward-ABI (5'-TGTAAAACGA CGGCCAGT-3 ') and Reverse-1 (5'-GGAAACAGCTATGACCATG-3') in addition to the internal primers Bc2F02 and Bc2R02 described above.

Comparison of the nucleotide sequences of genomic DNA and cDNA showed that the MIP102 gene contained two introns (FIG. 2) (73 bp (Intron 1) and 278 bp (Intron 2)). The positions of these introns were identical to those of AtKELP (Cormack et al., Homology). Southern blot hybridization using a 440 bp fragment of MIP102 cDNA was performed to estimate the number of genes encoding MIP102 homologs in B. campestris (FIG. 4). DNA probes were hybridized with one band in each lane and showed that MIP102 was encoded by a single copy gene in B. campestris .

Example 3 Binding Assay of a Deletion Mutation of MIP102

GST-fusion MIP102 with N- or C-terminal deletions was generated and used in protein-binding assays as described in Example 1 to determine MIP102 domains important for MP binding.

Plasmid pGEX-P-Bc2 was derived from phage clone MIP102 (described above). pGEX-Bc2 BamHI-EcoRI fragment (842 bp) was inserted between the BamHI and EcoRI sites of pGEX-6P-Bc2 (Amersham Pharmacia Biotech) to generate pGEX-P-Bc2.

To rescue pGEX-P-Bc2dN, the DNA fragment for the C-terminal half of MIP102 was amplified by PCR from pGEX-Bc2 using Bc2F02 primer (5'-AAYAARGARTTYGAYGAYGA-3 ') and pGEX5 primer (described above). And treated with T4 DNA polymerase and digested with NotI. The resulting 679-bp fragment was inserted between the SmaI and NotI sites of pGEX-6P-2.

DNA fragments for the N-terminal half of MIP102 for the structure of pGEX-P-Bc2dc were amplified by PCR from pGEX-Bc2 using pGEX1 primer (described above) and Bc2R05 primer (5'-GAGACTCGAGTCATCCCTCCTTAGCTCTTT -3 '). And digested with BamHI and XhoI. The resulting 331-bp fragment was inserted between the BamHI and XhoI sites of pGEX-6P-2.

pGEX-P-Bc2 was used for expression of GST-fusion MIP102 (GST-MIP102). pGEX-P-Bc2dN was used for GST-fusion MIP102 with deletion of N-terminal 86 amino acid residues (GST-MIP102dN), whereas pGEX-P-Bc2dC was GST-fusion MIP102 with deletion of C-terminal 79 amino acid residues Was used (GST-MIP102dC). The coding sequence derived from the PCR fragment was found to be free of any errors.

As shown in FIG. 5A, ³² P-labeled PKA-MP not only covers the full length (amino acids 1-165) (lane 1) of GST-MIP102, but also the N-terminal half (amino acids 1-86) (lane 3) of MIP102. Bound to GST-MIP102dC. The double band observed in lanes of GST-MIP102dC (FIG. 5B, lane 3) is due to protein degradation near the C-terminus; Lower bands with additional deletions showed binding activity. In contrast, no MP-binding activity was observed in GST-MIP102dN (lane 2), including GST (lane 4) and the C-terminal half (amino acids 87-165) of MIP102. These results suggest that the N-terminal half of MIP102 contains an MP binding domain.

Example 4 Inhibition of Cell-to-Cell Migration by the N-Terminal Region of MIP102

To enable visual observation of ToMV migration, the plasmid piL.G3 was used in which the coat protein (CP) gene of the viral genome was deleted and the green fluorescent protein (GFP) gene was inserted into the viral genome at the CP gene location. This plasmid was obtained from Tetsuo Meshi of Kyoto University (Tamai and Meshi, Mol. Plant-Microbe Interact. (2001) 14, 126-134). Plasmid pART7-Bc2dC-HA was also prepared to express the C-terminal deletion MIP102 (MIP102dC) obtained in Example 3. These methods of preparation are described below.

The plant expression binary vector pART7 was used to express MIP102dC (Adrew P. Gleave, Plant Molecular Biology (1992) 20, 1203-1207). DNA fragments of the N-terminal half of MIP102 include pGEX1 primer (described above) and Bc2R06HA primer (5'-TTGCTCTAGACTAAGCATAATCAGGAACATCATAAGGATATCCCTCCTTAGCTCTTTC-3 '); The Bc2R06HA primers (including the HA tag sequence) were amplified by PCR from pGEX-Bc2 and digested with SmaI and XbaI. The resulting about 350-bp fragment had a tag encoding the hemaglutinin epitope (HA) fragment sequence inserted at its C terminus. This fragment was inserted between the SmaI and XbaI sites downstream of the CaMV35S promoter of pART7 to generate pART7-Bc2dC-HA. Plasmid pART7-GUS expressing GUS was also prepared as a control plasmid.

Plasmids piL.G3 alone or both piL.G3 and pART7-Bc2dC-HA plasmids introduced these plasmids into tobacco ( N. benthamiana ) cells using a particle total method using leaf fragments of the actual leaves of tobacco. The introduction of genes into these cells by the particle gun method was performed using the BioRad transduction device PDS-1000 / He system according to the manufacturer's instructions. Green fluorescence was observed under fluorescence microscopy. When the viral genome is migrated to neighboring cells, green fluorescent proteins are produced in the cells containing the migrated viral genome, causing the cells to emit green light (FIG. 6, “multiple cells”). In contrast, in the absence of cell-to-cell migration of the virus, fluorescence occurs only in one cell (FIG. 6, “single cell”). Thus, virus migration can be detected according to fluorescence differences.

Next, the plasmid piL.G3 with GFP inserted in place of the CP gene while varying its proportions. And particle plasmids expressing the MP binding region of GUS (pART-GUS) or MIP102 (pART7-Bc2dC-HA) using leaf fragments of the actual leaves of tobacco were subjected to particle gun to introduce these plasmids into tobacco cells. The results are shown in Table 1.

Momentary gene introduction of N. benthamiana into real leaves by particle gun Plasmid combination DNA amount per introduction Fluorescence per half leaf ^a Multiple cell / total number ^b Only one cell light emission (part without virus migration) Multiple light emission (parts with virus migration) piL.G3 + pART-GUS (ToMV / GFP) (control) 1.0 μg: 2.5 μg 19 133 0.87 ± 0.094 piL.G3 + pART-Bc2dC-HA (ToMV / GFP) (MP binding region of MIP102) 1.0 μg: 2.5 μg 109 110 0.050 ± 0.11 0.5㎍: 2.5㎍ 47 46 0.49 ± 0.12

^a total number as a result of introduction into three to four leaves

^{b The} next number represents the standard deviation calculated from the results of introduction into 3-4 leaves.

The results shown in Table 1 show that the expansion of green fluorescence, a signal of viral genome migration, is inhibited by the introduction of pART7-Bc2dC-HA. Therefore, the expression of protein at the N terminus of intracellular MIP102 was considered to inhibit the function of viral MP and inhibit viral migration.

The ratio of cell-to-cell migration was observed with varying molar ratios of pART7-Bc2dC-HA and piL.G3. The results are shown in FIG. In Figure 7, the vertical axis represents the ratio of cell-to-cell migration, expressed as the fluorescence spot ratio (multiple cells / total number), while the horizontal axis represents the molar ratio of plasmid (pART7-Bc2dC-HA / piL.G3). As pART7-Bc2dC-HA increased relatively, the expansion of green light into multiple cells was reduced, i.e. inhibition of migration was recognized.

(Example 5) Effect of co-introduction of MIP102 expression plasmid when ER localized GFP expression plasmid was used

The rate of cell-to-cell migration was observed with varying molar ratios of pART7-Bc2dC-HA and piL.erG3 (ER localized GFP expression plasmid). piL.erG3 (ER localized GFP expression plasmid) was obtained from Tetsuo Meshi of Kyoto University (Jun Tami and Tetsuo Meshi, The Proceedings of Annual Meeting of Japanese Society of Plant Pathology (2000), Okayama University).

The results are shown in FIG. In Figure 8 the vertical axis represents the ratio of cell-to-cell migration, expressed as the fluorescence spot ratio (multiple cells / total number), while the horizontal axis represents the molar ratio of plasmid (pART7-Bc2dC-HA / piL.erG3). As pART7-Bc2dC-HA increased relatively, the expansion of green light into multiple cells was reduced, i.e. inhibition of migration was recognized. Comparison of Figures 8 and 7 showed that the degree of inhibition was greater when piL.erG3 (ER localized GFP expression plasmid) was used than when ER non-localized GFP expression plasmid (piL.G3) was used.

Example 6 Production of Transformed Plant Using Plasmid and Structure of Plasmid for Producing Transformed Plant by Introduction of MIP Gene

Phage DNA isolated from the MIP102 clone to generate transformed plants was used to rescue the expression plasmid pART27-Bc2dC-HA.a, which produces the N-terminal binding region of MIP102. A 2.45-kb NotI fragment derived from pART7-Bc2dC-HA (described above) was inserted at the NotI site of pART27 (Adrew P. Gleave, Plant Molecular Biology (1992) 20, 1203-1207) pART27-Bc2dC-HA. a was generated. The structural diagram of this vector is shown in FIG.

pART27-Bc2dC-HA.a was introduced into the leaf pieces of tobacco by the Agrobacterium method. Agrobacterium methods are described in Nagel et al. (Microbiol. Lett., 67, 325 (1990)). The above-described expression vectors were used to transform Agrobacterium by electroporation. The transformed Agrobacterium was then introduced into plant tissue by the leaf disk method (Rabo-Manyuru [Laboratory Maunal], Syokubutsu-Idenshi-no-Kino-Kaiseki [Fenctional Analysis of Plant Gene] Masaki Iwabuchi and Toshiro Shimura, eds., 1992, Maruzen, pp. 31-56). Every two weeks the above-described leaf fragments were secondary cultured and transformed based on the presence of kanamycin resistance due to the expression of kanamycin resistance genes derived from pART27 introduced into tobacco cells along the above-described polynucleotides. Screened for tobacco cells. Selected transformed tobacco cells were re-differentiated into plants.

Transformed tobacco leaves with MIP102 introduced were ground in liquid nitrogen and extracted at 100 ° C. for 4 minutes in SDS-PAGE sample buffer. Extracts were SDS-polyagrylamide electrophoresis (SDS-PAGE, polyacrylamide gel concentration 10-20%) and anti-KELP antibody (pGEX-P-KELP (produced is described in Example 9 below)) as a probe. Produced by immunizing rabbits with purified KELP derived from) and anti-HA antibodies (obtained from MBL) were subjected to Western blotting analysis by commonly used methods.

The results are shown in FIG. (A) Coomassie Brilliant Blue (CBB) -stained gel, (B) results of use of HA tag antibody and (C) results of use of KELP antibody. The leftmost lane of each resulting lane is the molecular weight marker and the remaining lanes show the transformed tobacco strains 101, 102, 105 and 117 in order from the left. The rightmost lane represents non-transformed tobacco (control). In these results, specific bands of 14 kDa, believed to correspond to MIP102, were detected in 101, 103 and 118, but not in transformed tobacco strain 105 and non-transformed tobacco (control).

Example 7 Investigation of Virus Cell-to-Cell Migration Due to ToMV-ΔCP / GFP Transcription Infection

Transformed tobacco strains with MIP102, 101, 103, 105, and 118 introduced with ToMV-ΔCP / GFP transcription products (RNA) to observe viral migration in transgenic tobacco with introduced MIP102, their regeneration To the leaves of plants 101r, 103r, 105r and 118r and non-transformed plants (SR1). It should be noted that regenerated plants are produced from leaf fragments according to methods commonly used. Application of the transcript was carried out as follows. pTL.G3 (obtained from Tetsuo Meshi of Kyoto University; in this plasmid the T7 promoter was transcribed upstream of the viral gene instead of the CaMV 35S promoter of piL.G3) was treated with T7 RNA polymerase to obtain RNA transcripts. 1 μg of RAN transcript was inoculated into tobacco leaves with a width of 3-5 cm. After 2 days the infected leaves were cut and observed with a fluorescence microscope to observe fluorescence from GFP. The results are shown in Table 2.

Infection of ToMV-ΔCP / GFP Transcripts with Transformed Tobacco Transformed Tobacco Populations Number of fluorescent spots Transformed Tobacco Populations Number of fluorescent spots One cell Multiple cells One cell Multiple cells 101 0 0 101r 0 0 103 0 0 103r 0 0 105 0 51 105r 0 3 118 0 0 118r 0 0 101, 103 and 118 are MIP120dC high expressing plants. Non-Transformer 1 0 0 Non-transformer 2 0 6 Transformed Tobacco Populations Number of fluorescent spots Number of fluorescent spots 101r, 103r, 105r and 118r are regenerated from 101, 103, 105 and 118. One cell Multiple cells One cell Multiple cells 101r 0 One 0 4 103r 0 0 0 0 Non-Transformer 1 0 48 0 45 Non-transformer 2 0 108 0 108

As shown in Table 2, MIP102 was expressed very high in transformants 101, 103 and 118 of Example 6 and their regenerators 101r, 103r and 118r and the expansion of green fluorescence was inhibited in these plants. Expansion of green fluorescence was observed in plants 105, its regenerating 105r and non-transformed plants (control) without high expression. Thus, virus migration was shown to be inhibited by plant expression MIP102.

Example 8 Investigation of Virus Infection by ToMV Particle Infection

Viral particle infection was performed to investigate whether resistance to ToMV infection was obtained by transformed tobacco with MIP102 introduced and accumulation of viral coat protein (CP) in infected and upper leaves was investigated. Regenerated 101r, 103r, 105r and 118r of transformed tobacco 101, 103, 105 and 118 with introduced MIP102 and leaves of non-transformed plants (control) were infected with 0.06 μg ToMV virus particles. After 10 days the infected leaves and the leaves in the upper position (top leaves) were cut, crushed in liquid nitrogen and extracted for 4 minutes at 100 ° C. in SDS-PAGE sample buffer. Extracts were subjected to SDS-polyacrylamide electrophoresis (SDS-PAGE, polyacrylamide concentration was 10-20%) before gel staining with Comassie Brilliant Blue.

The results are shown in FIG. Opposite end lanes represent molecular weight markers and the remaining lanes are transgenic tobacco strains 101r, 103r, 105r and 118r (which are regenerated from transformed tobacco strains 101, 103, 105 and 118) in order from the left Tobacco (SR1). For each strain the left lane represents the upper leaf (U) while the right lane represents the infected leaf (I). The bands indicated by the arrows correspond to coat proteins. This band was significantly observed in infected and upper leaves of non-transformant SR1 and in transform 105r which did not show high levels of expression of MIP102 in Example 6. Accumulation of coat protein was reduced and virus resistance was predicted in transformed strains 101r, 103r and 118r where MIP102 was highly expressed and inhibition of virus migration was confirmed.

Example 9 Binding Essay with AtKELP

CDNA for AtKELP was isolated from the A. thaliana cDNA library to investigate whether ToMV MP binds to AtKELP with high levels of homology (75%) to MIP102. The plasmid encoding GST-fusion AtKELP (GST-AtKELP) was rescued and the purified fusion protein was used for ToMV MP and protein-binding assays as probes. Protein-binding assays were performed as described in Example 3.

AtKELP expression plasmids were constructed as follows. The coding sequence of AtKELP (Cormack et al., Homology) was A. thaliana 5 'by PCR with KELP-F01 primer (5'-ACAGGAATTCCTAAAAATGGAGAAAGAGA-3') and KELP-R01 primer (5'-TTGCCTCGAGTCAGACACGCGATTCCATTT-3 '). -Amplified from stretch cDNA library (Clontech). The amplified 0.52-kb fragment was digested with EcoRI and XhoI and inserted between EcoRI and XhoI sites of pGEX-6P-1 to generate pGEX-P-KELP. The coding sequence of KELP was found to be without any base change. To add the phosphorylation sequence of protein kinase A, Hanhan oligonucleotides KD-PKA1 (5'-GATCACGTCGTGCATCTGTTG-3 ') and KD-PKA2 (5'-GATCCAACAGATGCACGACGT-3') were annealed and incorporated into the BamHI site of pGEX-6P-1 3 sets of oligonucleotides inserted to rescue pGEX-6P1-3PKA inserted in the appropriate orientation downstream of the GST ORF. For the structure of pGEX-P-3xPKA-KELP, a 0.52-kb EcoRI-XhoI fragment of pGEX-P-KELP was cloned between the EcoRI and XhoI sites of pGEX-6P1-3PKA. pGEX-P-KELP and pGEX-P-3xPKA-KELP were used for expression of GST-AtKELP and GST-PKA-AtKELP, respectively.

As shown in FIG. 12, GST-AtKELP (lane 1) and positive control GST-MIP102 (lane 2) showed MP-binding activity while negative control GST did not.

Example 10 Combined Essay of MPs of CTMV-W and CMV

We also measured the binding activity of MIP102 and AtKELP on MPs of other plant viruses. In this case MPs from CTMV-W and CMV were used. Protein binding assays were performed as described in Example 3.

CTMV-W MP expression plasmids were performed as follows. Plasmid pTW62 (Shimamoto et al., (1998) Arch. Virol. 143, 1801-1813) with cDNA from wasabi strain (CTMV-W) of Crucifer tobamovirus was digested with SalI. The resulting 1.4-kb fragment, including the coordinating sequence of MP, was inserted into the SalI site of pAS2-1 (Clontech) in the same direction as the Gal4 gene. The resulting plasmid was digested with NcoI and BamHI and treated with Klenow fragments and self-ligated to produce pAS-W30K with an ORF of MP continuous to Gal4. The 1.4-kb BamHI-NotI fragment of this plasmid was inserted between BamHI and NotI sites of pGEX-5X-3 (Amersham Pharmacia Biotech) to generate pGEX-W30K. PGEX-W30K was further digested with BamHI to adjust the CTMV-W MP reading frame with GST, treated with Klenow and self-ligated, pGEX-W30KfB used for expression of GST-fused CTMV-W MP (GST-CTMMP). Generated.

CMV MP expression plasmids were performed as follows. Plasmid pUCMV03 (Hayakawa et al., (1989) J. Gen. Virol. 70, 499-504) containing RNA 3 cDNA derived from O strain of CMV (CMV-O) was digested with AvaI and treated with Klenow fragments. Digestion with SalI separated the 1.2-kb AvaI (blunted) -SalI fragment. These fragments, including 3a ORF (MP), were inserted between the SmaI and XhoI sites of pGEX-6P-1 to rescue pGEX-P-CMVOMP encoding GST-fusion CMV MP (GST-CMVMP).

As shown in FIG. 13, ³² P-labeled PKA AtKELP was bound to ToMV, CTMV-W and CMV-O. Similar binding activity was observed when ³² P-labeled MIP102 was used as a probe (data not shown).

The above-described embodiments exemplarily illustrate various aspects of the invention and how oligonucleotides of the invention are produced and used. The present invention is not limited thereto.

Polynucleotides capable of imparting viral resistance that can be used for plant breeding and methods of imparting viral resistance to plants using the polynucleotides thereof are provided. In addition, polynucleotides are introduced to provide plants endowed with resistance to plant viruses.

Claims (9)

A method of imparting resistance to a plant virus to a plant consisting of introducing a polynucleotide encoding a protein capable of being expressed in the plant into a cell of the plant and interacting with a mobile protein of the plant virus The protein of claim 1, wherein the protein encoded by the polynucleotide is represented by positions 1 to 86 of SEQ ID NO: 2, comprising a sequence or comprising a sequence having one or several amino acid substitutions, deletions, and / or additions, Characterized in that it binds to the mobile protein of plant viruses The method of claim 1, wherein the polynucleotide comprises a nucleotide sequence capable of hybridizing to the sequence shown in positions 14 to 271 of SEQ ID NO: 1 or the nucleotide sequence under stringent conditions. The method of claim 1, wherein the plant virus is Tobamovirus The method of claim 1, wherein the plant virus is tomato mosaic virus (ToMV). The method of claim 1, wherein the polynucleotide is derived from Brassica campestris or Arabidopsis thaliana . Plants produced by the method according to claim 1 8. The plant according to claim 7, wherein the plant is a monocotyledonous plant or a dicotyledonous plant. 9. A plant according to claim 8, wherein the plant is tobacco.